Antibiotic resistance is a major problem in clinical settings as bacteria continuously evolve to circumvent drugs. The majority of our understanding on antibiotic resistance comes from studies on the genetic changes that cause it, however bacteria can also transiently survive antibiotic exposure even without permanent genetic changes. A key question is whether transient resistance can serve as a stepping stone to permanent drug resistance. To address this, we focus here on multi-drug efflux pumps, which are a canonical example of a transient resistance mechanism. Expression of multi-drug efflux pumps, such as AcrAB-TolC and its homologs, allows cells to export a broad range of antibiotics including b-lactams, fluoroquinolones, tetracyclines, and many others. Although pumps enable survival in antibiotics, they are costly to express so cells commonly turn them on only temporarily. Two recent studies have shown that in Escherichia coli AcrAB-TolC expression varies from cell to cell within a population and that pump levels and drug export rates vary within populations as a result. Importantly, our preliminary data show a link between single-cell efflux pump expression and mutation rate. Based on these results, our central hypothesis is that the AcrAB-TolC pump provides transient resistance and elevated mutation rates that differ between cells in a population, allowing some cells to survive antibiotics longer while increasing mutagenesis to promote the emergence of resistant mutants. We will test this hypothesis using an approach that integrates single-cell time-lapse microscopy, optogenetically-controlled efflux pumps, and mathematical modeling to measure the duration and time dependence of efflux-mediated antibiotic survival and mutation. Our approach centers around two Aims: (1) Quantify single-cell relationship between AcrAB-TolC expression and duration of transient resistance. (2) Determine how variability in AcrAB- TolC impacts expression of mutation-related genes and the emergence of permanent resistance. This research is significant because multi-drug efflux pumps are ubiquitous in pathogenic bacteria and are often implicated in the initial stages of drug resistance. In addition to measuring the role of efflux pumps in the evolution of antibiotic resistance, this work is likely to be generally relevant for understanding and eliminating nucleation points for drug resistance. The work is innovative because of the dynamic, single-cell approach to quantifying the emergence of antibiotic resistance and in addition proposes a novel role for efflux pumps in elevating mutation rates.